Loss of vascular homeostasis is a major contributor to the morbidity and mortality of diabetes, and treatments that restore or prevent loss of vascular homeostasis would be of immense value to diabetic patients. However, the processes leading to loss of vascular homeostasis are poorly understood, and effective treatments are not available. The long term goal is to better understand mechanisms underlying the loss of vascular homeostasis in diabetes in order to develop preventive and therapeutic protocols. Dysregulation of the 268 proteasome has been implicated in the loss of vascular homeostasis in diabetes. It appears to involve enhanced 268 proteasome functionality resulting in down regulation of nitric oxide (NO). The preliminary data suggests that NO itself regulates 268 proteasomes, a mechanism that might be compromised in diabetes. Therefore, the objective in this application is to identify how NO regulates 268 proteasomes. The central hypothesis is that eNOS-derived homeostatic levels of nitric oxide, via maintaining the 0-GlcNAc modification of the regulatory complex PA700 (Rpt2), functions as an endogenous inhibitor of 268 proteasomes to preserve vascular homeostasis, which is lost in diabetes. The rationale for the proposed research is that the identified mechanism may help to understand how vascular homeostasis is lost in patients of diabetes, epidemic of which is believed to hit half of this country by 2020. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) Determine if endothelial NO inhibits 268 proteasome functionality via maintaining 0-GlcNAc modification of the regulatory complex PA700 (Rpt2); and 2): Determine if pharmacologic or genetic strategies that restore eNOS-derived NO or enhance PA700 (Rpt2) 0-GlcNAc modification suppress 268 proteasome functionality and mitigate endothelial dysfunction in diabetes. The approach is innovative, because it utilizes a proteasome reporter system to study diabetic vascular endothelial function. The proposed research is also significant, because it is expected to provide insight into therapeutic interventions in addition to fundamentally advancing the fields of vascular medicine.